ENHANCED DEGRADATION AND PEPTIDE SPECIFICITY OF MMP-SENSITIVE SCAFFOLDS FOR NEOVASCULARIZATION OF ENGINEERED TISSUES
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Biomaterial strategies for engineering tissues of clinically relevant size require the formation of rapid and stable neovascularization. The ability of an engineered scaffold to induce vascularization is highly dependent on its rate of degradation. During the process of material degradation, the scaffold should degrade in a manner allowing for cellular infiltration, lumen formation, and extracellular matrix (ECM) synthesis. Matrix metalloproteinases (MMPs) play a key role in mediating cell-induced proteolytic matrix degradation, remodeling, and controlled neovascularization. Poly (ethylene glycol) PEG hydrogels have been extensively investigated as scaffolds for tissue engineering applications due to their ease of chemical modification allowing for the recapitulation of key aspects of the neovascularization process. The goal of the work described in this thesis was to develop strategies to enhance and control the degradation of MMP-sensitive PEG diacrylate (PEGDA) hydrogels without inducing changes to the bulk physical and mechanical properties of the material and to further study the effect of the cleavage site concentration and MMP-sensitive peptide substrate specificity on the rate of neovascularization and tissue remodeling in vitro and in vivo. In the first part of this study, a detailed investigation was completed to investigate the effects of the mechanical and physical properties of the scaffolds as well as the role of proteolytically mediated hydrogel degradation on 3D fibroblast invasion within MMPsensitive PEGDA hydrogels. Initial studies focused on the use of a modified version of a previously published multistep conjugation method to generate degradable PEGDA macromer conjugates containing variations in the number of MMP-sensitive domains. Theoretical and experimental characterization of this multistep conjugation demonstrated xi that this method leads to the formation of multiple species that directly affect the compressive modulus and degradation rate of the scaffold making it difficult to control degradation independent of alterations in the bulk physical and mechanical hydrogel properties. After manipulation of multiple polymerization conditions, hydrogels with similar compressive moduli but different hydrogel degradation rates were synthesized. These initial studies showed that an increase in the incorporation of proteolytically sensitive domains in PEGDA hydrogels of similar modulus lead to enhanced degradation and 3D fibroblast invasion. In this study, the role of soluble FGF-1 on fibroblast invasion within these scaffolds was investigated and it was demonstrated that the inclusion of FGF-1 in the scaffolds results in further enhancement of fibroblast invasion in a dosedependent fashion. Further studies were necessary to develop a more controllable and robust approach in tuning scaffold degradation independent of alterations in the bulk physical and mechanical properties. In order to address this, a novel approach was developed to engineer protease-sensitive peptides with multiple proteolytic cleavage sites that could be covalently crosslinked into hydrogels without compromising the physical and mechanical biomaterial properties. This approach avoided the need for utilizing a multistep conjugation process as peptides could be incorporated into the backbone of PEG using a single step conjugation. Using this approach, hydrogels formed with the engineered peptides led to significantly enhanced degradation and neovascularization in vitro as compared to scaffolds with a single protease sensitive peptide between crosslinks. In addition, hydrogels with enhanced susceptibility to degradation promoted vascularization over a wider range of matrix properties. This approach allowed for controlled xii concentration of the proteolytic cleavage sites within the matrix and thus tuning of hydrogel degradation for tissue engineering applications. In the final study, MMP-sensitive peptide substrates specific to degradation by MMPs known to be expressed during neovascularization were screened for degradation and their role in neovascularization. MMP-sensitive PEGDA hydrogels (SSite and TriSite) were synthesized with peptide substrates sensitive to cleavage by MMP-2, MMP- 9, MMP-14, a mixed sequence of MMP-2, 9 and 14, and compared to the peptide substrate used in the previous studies, which is degraded by collagenase enzymes. The hydrogels were evaluated for their sensitivity and specificity to degradation by MMPs, in terms of cleavage site concentration, and for their role in neovascularization and tissue remodeling in vitro and in vivo. The presented approach allows for the incorporation of varying cleavage site concentration and MMP-sensitive peptide substrates into PEG hydrogels without alterations in the mechanical and physical properties of the hydrogels. Results showed that without the incorporation of growth factors in this scaffold, vascularization and tissue invasion was supported in all MMP-sensitive hydrogel groups regardless of the MMP-sensitive peptide substrate embedded in the matrix. In addition, the cleavage site concentration had a profound impact in enhancing vascularization in vitro and tissue invasion in vivo. These techniques can be used to tune the properties of polymer scaffolds for neovascularization and tissue remodeling. In addition, these studies provide insight into the effect of the physical, mechanical, and degradative properties of these systems and on the role of cleavage site concentration, and MMP substrate specificity on xiii neovascularization and tissue invasion within proteolytically degradable PEG hydrogel constructs.